Industrial process for food liquids decontamination from chemical and/or biological contaminants

ABSTRACT

Procedure for the decontamination of a food liquid from one or more chemical and/or biological contaminants, based on the contact of said liquid with at least a biocompatible membrane, to which antibodies specific for said contaminants are covalently bound.

PRIOR ART

Food liquids, e.g. wine, milk, fruit and vegetables juices, beer, andwater may contain chemical or biological contaminants.

Out of the chemical contaminants, the most common are:

-   -   parasiticides, weed-killers, pesticides: they may reach the food        from the soil through the fruit or vegetables used as starting        materials or, as concerns milk, through the aliments introduced        with the diet in the producing animal;    -   drugs, hormones and metabolites thereof: they are originated, in        the milk production cycle, from the uncontrolled treatment of        the animal or, as concerns hormones, from the physiological        period of milking;    -   process contaminants: they derive e.g. from the malolactic        fermentation of wine.

The most frequent biological contaminants are toxins, e.g. of bacterialor mycotic origin, which may reach the food through the fruit orvegetables used as starting materials or may be generated during thefood liquid preparation process.

The amount of the possible contaminations and difficulty in monitoringthe events at their origin make the problem of food liquidscontamination of topical interest as it is of paramount importance forthe control of the consumers' risk factors.

For example, the nephrotoxicity induced by mycotic toxins is well known(I. Baudrimont, A. M. Betbeder, A. Gharbi, A. Pfohl-Leszkowicz, G.Dirheimer and E. E. Creppy, Effect of superoxide dismutase and catalaseon the nephrotoxicity induced by administration of ochratoxin A in rats,Toxicology, 1994, 89(2), 101). So are the problems entailed by thepharmacoresistance following an uncontrolled use of antibiotics, and theeffects (especially on populations at risk) of the presence ofpharmacologically active metabolites, such as glucocorticoids andbiogenic amines in food.

Therefore, there is an urgent need for techniques allowing a totaldecontamination of food liquids from all aforementioned contaminants.

So far, said decontamination has been carried out by techniquesexploiting the physical adsorption of contaminants by inert substrates,such as active carbon, gel, cellulose and derivatives thereof. However,the use of said techniques is limited as it effects an insufficientdecontamination and, being based on nonspecific physical processes,removes substances, e.g. pigments, flavouring agents or even nutrients,which substantially determine the food primary characteristics.

Patent application M199A002622 by the Applicant describes an innovativeand ameliorative decontamination technique envisaging the complexation(elimination) of the toxic contaminants present in the food liquid bythe corresponding insolubilised specific polyclonal antibodies.

In particular, immunoglubulins specific for the contaminant to beeliminated are insolubilised by adhesion to glass or plasticmicrospheres or to magnetised metal microspheres, optionally coated withchemically derivatizable polymers and added to the contaminated liquidin precise and predetermined molar concentration ratios. Afterincubation, the toxic residues-immunoglobulins complexes that form areeliminated by filtration.

However, some drawbacks are inherent in the industrial application ofsaid technique:

-   a. the microspheres used as a means of immobilisation and    utilisation of decontaminants, are precipitable. Therefore, the food    liquid must be vigorously stirred. It follows that the process,    which, moreover, is not always technically applicable, involves    considerable modification costs;-   b. since the surface of contact between the immunoglobulins and the    liquid to be decontaminated is small, the decontamination time is    relatively long and not always compatible with the production    processes;-   c. since the antibody is bound to the solid support by adhesion,    i.e. through a weak bond, it tends to be detached therefrom in    considerable amounts during the washing and reactivation steps,    carried out to allow its use in successive processes. It follows    that it cannot be reused as many times as needed not to    significantly affect the production costs;-   d. the liquid filtration required at the end of the process    determines an increase in production times and costs.

Substrates of a different nature, e.g. membranes obtained innitrocellulose or other physically reactive polymers, wheretoimmunoglobulins are bound by adhesion, are known and especially used inclinical biochemistry for diagnostic purposes.

Recently, they have been also used in the detection of mycotoxins infoods and biological fluids. By way of example, mention is made of:

-   E. Usleber, E. Schneider and G. Terplan, Direct enzyme immunoassay    in microtitration plate and test strip format for the detection of    saxitoxin in shellfish, Left. in Appl. Microbiol., 1991, 13, 275.-   S. De Saeger and C. Van. Peteghem, Dipstick Enzyme Immunoassay to    Detect Fusarium T-2 Toxin in Weath, Appl. Environ. Microbiol., 1996,    62(6), 1880.-   E. Usleber, E. Schneider, G. Terplan and M. V. Laycock, Two formats    of enzyme immunoassay for the detection of saxitoxin and other    paralytic shellfish poisoning toxins, Food Additiv. and    Contaminants, 1995, 12 (3), 405.

SUMMARY

It has surprisingly been found that biocompatible membranes, preferablyof a polymeric nature, to which antibodies specific for contaminants arecovalently bound, are advantageously used in the decontamination of foodliquids. They allow to solve the problems entailed by thedecontamination techniques known in the art and bring about surprisingresults in terms of efficiency and application simplicity.

Therefore, the present invention refers to a new procedure for thedecontamination of food liquids, based on the use of the aforesaidmembranes.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide a process for foodliquid decontamination from one or more chemical and/or biologicalcontaminants, which consists in the contact of said liquid with at leasta biocompatible membrane, to whose surface antibodies specific for saidcontaminants are covalently bound.

It is a further object of the present invention to provide a membranefor the decontamination of food liquids from chemical and/or biologicalcontaminants, to be used in the process of the invention. Said membraneconsists of a biocompatible material and is characterised by the factthat antibodies specific for said contaminants are covalently bound toit or to its surface.

According to a particularly preferred embodiment of the invention, themembranes, preferably in the form of strips, are immersed in the liquidto be decontaminated. Said strips are kept taut in the liquid by somefloats, e.g. hollow plastic balls, at one end and by some balanceweights at the other end. The membranes are kept immersed in the liquidto be decontaminated for a period preferably ranging from 1 to 24 hrs,depending on the contaminant concentration, on the temperature and onthe presence or absence of stirring. The liquid decontamination is morerapid under stirring. According to the present invention, the foodliquid can be completely and rapidly decontaminated also withoutstirring. This is particularly advantageous when the stirring of theliquid mass to be decontaminated is unadvisable or involves excessivecosts.

Furthermore, the decontamination is more rapid at room temperature thanat temperatures below room temperature. In fact, at room temperature,the decontamination preferably takes from 1 to 6 hrs.

Once the treatment has been completed, the membrane/s of the inventionis/are separated from the liquid by simple removal, i.e. without theseparation procedures, such as filtration, required by techniques knownin the art. This brings about considerable advantages in terms of costand safety.

The food liquids that can be decontaminated by the procedure accordingto the present invention are, e.g. wine, milk, fruit and vegetablesjuices, beer and water.

According to a particularly preferred embodiment of the presentinvention, the membrane/s consists/consist of a biocompatible polymerthat is chemically conjugated with antibodies. The biocompatible polymeris synthetic, semi-synthetic or natural and is suitable for thepreparation of a membrane with a mechanical resistance sufficient forthe use of same in the claimed procedure.

Membranes are preferably in the form of woven or non-woven fabric

The polymer is preferably selected from the group consisting of nylonand derivatives thereof, cellulose derivatives and polyacrylates,nitrocellulose and nylon 66 being particularly preferred. Membranes canconsist of only one of these materials or being prepared with a mixtureof them (e.g. alcantara consists in 60% polyester and 40% polyurethane).Antibodies specific for the contaminants to be eliminated areimmobilised on the membranes of the invention. Preferably, saidantibodies are polyclonal antibodies, obtained by immunisation ofmedium-large sized animals according to methods known in the art (cf. A.Johnstone and Thorpe, Immunochemistry in Practice, 1982, 27-30,Blackwell Sci. Publ., Oxford).

The antibodies are immobilised on the membranes of the invention bychemical conjugation reactions known in the art, specifically optimisedfor that purpose.

The chemical conjugation with the antibody preferably occurs through alinker, whose function is to reduce the steric hindrance and,consequently, favour the antibody conjugation with the membrane and theinteraction between the antibody and the contaminant/s. Furthermore, theconjugation through a linker increases the density of the antibodies onthe membrane surface. These properties allow a more efficientdecontamination.

Diamino-monocarboxylic amino acids or monoamino-dicarboxylic amino acidscan be used as linker, particularly Arginine, Lysine, Aspartic Acid andGlutamic Acid.

Another particularly preferred linker is the following:—CH2-CH2-SO2-CH2-CH2-N H—(CH2)4-N═CH—(CH2)3-CH═O

The process of the invention allows the elimination from a liquid foodof any contaminant against which specific antibodies can be produced.

In particular, the aforesaid contaminants may be either of a chemicalnature, such as for example parasiticides, weed-killers, pesticides,drugs and metabolites thereof, hormones and metabolites thereof, andundesired substances that originate during the food liquid productionprocess, or of a biological nature, such as for example toxins.

In particular, as described in detail in the experimental part thatfollows, the procedure of the invention is very effective for theelimination of the following contaminants from food liquids:

-   -   atrazine, aflatoxin, ochratoxin, fumonisine, cadaverine,        putresceine, urethane, progesterone and inactivated salmonella.

The process according to the present invention, which uses membraneswhereto antibodies directed against the various contaminants are bound,allows the decontamination of a liquid from several contaminants in asingle operation.

Therefore, in the decontamination procedure according to the invention,one or more membranes per contaminant is/are used.

The total surface of the membrane/s per contaminant is such that themolar ratio of the immobilised antibody to the contaminant recognized bythe antibody is preferably ≧1. According to a particularly preferredembodiment, said ratio ranges from 1 to 5 and preferably from 1 to 2.

Compared with the techniques for food liquid decontamination known inthe art, the procedure of the invention offers many and unexpectedadvantages.

In particular, as demonstrated by the following examples, the procedureof the invention is of easier application since it does not require anystirring of the liquid. Furthermore, said procedure brings about acomplete decontamination within a much shorter time than necessary forthe procedure described in patent application MI99A002622, which—for thedecontamination—utilises antibodies immobilised on microspheres. Asshown in more details in Example 18, a further advantage of the presentprocedure is that the claimed membranes may be regenerated bycontaminant removal by washing, e.g. with 0.1 N HCl, and may be re-usedin successive decontamination procedures, without losing theirdecontamination power. That brings about considerable advantages interms of process costs.

EXAMPLE 1 Preparation of atrazine-BSA (Bovine Serum Albumin) conjugate

1. Diazoderivative Preparation

10 mg Atrazine (4.6×10⁻⁶ mol) was added with 10 μl 1N HCl (5×10 mol).The resulting mixture was brought to the desired consistency by means ofa spatula, poured into a test tube, which was placed into boiling water,and added with 900 μl distilled water. The resulting atrazine.HClsolution was added with 0.5 ml 1N HCl and cooled in an ice bath. Once 1mg NaBr was added, 260 μl NaNO₂ cold solution in a concentration of 1mg/ml (260 mg; 4.4×10⁻⁶ mol) was added dropwise under stirring. Stirringwas continued for 1 hr in ice to give the captioned diazoderivative.

2. Conjugation with BSA

A BSA solution in a concentration of 8.4 mg/ml in 0.1M borate buffer, pH9, was added dropwise under stirring over a period of 15 min with thediazoderivative solution prepared as per point 1; the pH was maintainedconstant by addition of 1 NaOH. The mixture was caused to react in icefor 2 hrs and dialysed against PBS (Phosphate Buffered Saline).

EXAMPLE 2 Preparation of aflatoxin-BSA conjugate

1. Preparation of Benzidine bis-diazoderivative

Benzidine.2HCl (26 mg) was dissolved in 4.5 ml 0.2N HCl, added with 18mg NaNO₂ dissolved in 0.5 ml distilled water. The reaction was carriedout in an ice bath under stirring for 1 hr; an orange colour immediatelydeveloped.

2. Conjugation with BSA

A BSA solution (50 μl) in a concentration of 10 mg/ml in 0.16M boratebuffer-1.3M NaCl, pH=9, was added with 50 μg lyophilised aflatoxin. Theresulting mixture was added with a mixture consisting of 17 μlbis-diazo-benzidine solution as per point 1 and 33 μl borate buffer. Thecolour turned brown and the reaction was continued at 4° C. for a periodof 2 hrs under desultory stirring. The reaction mixture was dialysedagainst PBS.

EXAMPLE 3 Preparation of ochratoxin-BSA conjugate

A BSA solution (30 ml) in a concentration of 10 mg/ml in 0.1M acetatebuffer, pH=5.5, was added with 9.32 ml ochratoxin solution in aconcentration of 5 mg/ml in 9/1 water/methanol. The mixture was addedwith 750 mg EDAC (Sigma E1769) and caused to react at room temperaturefor a period of 2 hrs; during the first 30 min, the pH was controlledevery 5 min.

The reaction mixture was dialysed against PBS, pH 7.4, in 10 Kd cut-offtubing.

The conjugation ratio of BSA to ochratoxin was 1:25 by mmoles.

EXAMPLE 4 Preparation of fumonisine-BSA conjugate

1. Preparation of Fumonisine B1-glutaraldehyde-BSA

A solution of BSA (Sigma A7906) (15 ml) containing 1 mg/ml protein in0.1M PBS, pH 7.4, was dialysed at 4° C. overnight against 200 ml 0.2%glutaraldehyde solution (Sigma G5882). The activated BSA solution wasfurther dialysed against 500 ml PBS (three changes min) to remove excessunreacted glutaraldehyde.

The activated BSA solution was added with 25 mg fumonisine B1 (SigmaF1147) dissolved in 1 ml 0.06M DMSO. The resulting mixture was caused toreact under stirring at laboratory temperature for 3 hrs and then at 4°C. overnight, and finally dialysed against PBS.

The conjugate obtained consisted of 0.93 mg BSA and 0.15 mg fumonisineB1/ml.

2. Preparation of fumonisine B1-CDI-BSA

A BSA solution (Sigma A7906) (15 ml) containing 1 mg/ml protein in 0.1Macetate buffer, pH 6.5, was prepared. The mixture was added with 2.5 mgfumonisine B1 dissolved in 1 ml of the said buffer containing DMSO inthe final concentration of 0.06 M and, under stirring, with 25 mgpowdered carbodiimide (CDI) (Sigma E1769). Stirring was continued atlaboratory temperature for 1 hr, while the pH was controlled every 10min and adjusted to 0.5 as long as necessary. The mixture was caused toreact at 4° C. overnight and successively dialysed against PBS to removeexcess unreacted reagents.

A conjugate containing 0.93 mg BSA and 0.15 mg fumonisine: B1/ml wasobtained.

EXAMPLE 5 Preparation of cadaverine-azo-BSA conjugate

-   Cadaverine: 1,5-diaminopentene [H₂N—(CH₂)₅—NH₂]-   Conjugate: H₂N—(CH₂)₅—N═N-BSA    1. Preparation of cadaverine mono-diazoderivative

The reaction was performed in an ice bath.

Cadaverine dihydrochloride (35 mg; 0.2 mmol)) was dissolved in 5 mlwater containing 0.3 mmol HCl and 4 mg NaBr (0.04 mmol).

The solution was slowly added over a period of approx. 10 min, understirring, with 14.5 mg (0.21 mmol) NaNO₂ dissolved in 1 ml icy water.

Once the presence of excess HNO₂ was checked with iodine-starch treatedpaper, the reaction was continued for further 10 min to give[NH₂—(CH₂)₅—N═N—OH].

2. Preparation of cadaverine-azo-BSA conjugate

A BSA solution (10 ml) in a concentration of 10 mg/ml in 0.1M boratebuffer, pH=9.0, containing 0.13M NaCl was added, under stirring and inan ice bath, with 2 ml cadaverine diazoderivative solution prepared asdescribed above.

The resulting solution was caused to react under desultory stirring at4° C. for 2 hrs and then dialysed against PBS in 10 Kd cut-off tubing.The pH was then adjusted to 9.0.

The cadaverine/BSA conjugation ratio obtained was 23.5:1.

EXAMPLE 6 Preparation of putresceine-azo-BSA conjugate

The putresceine-azo-BSA immunogen was synthesized as described inExample 6, in the same reaction molar ratios (by multiplying the amountindicated for cadaverine by 0.92).

Owing to structural analogy, the antibodies produced against cadaverinereacted against putresceine to a sufficient extent to bring about thecomplexation of same.

EXAMPLE 7 Preparation of ethyl-carbamate-N═N-BSA conjugate

1. Ethylcarbamate (Urethane) Diazotisation.

Urethane (35.6 mg; 0.4 mol) was dissolved in ml 0.1N HCl. The solutionwas cooled in Ice, added under stirring with 24.8 mg NaNO₂ dissolved in2 ml icy water under stirring. Stirring was continued for 1 hr in ice.

2. Formation of the Conjugate with BSA

A solution containing 130 mg BSA in 13 ml 0.16M borate buffer-0.13MNaCl, pH=9.0, was cooled in ice and slowly added under stirring with a 1ml diazo-ethyl-carbamate solution. The resulting solution was caused toreact for 2 hrs and then dialysed against the same buffer in 10 kDcut-off tubing.

The ethylcarbamate/BSA ratio obtained was 40:1 by mol.

EXAMPLE 8 Preparation of progesterone-BSA conjugate

1. Progesterone Activation

Progesterone-3(O-carboxymethyl)oxime (60 mg; 0.15 mM) (Sigma P3277) and37.5 μl (corresponding to 0.15 mM) tri-n-butylamine were dissolved in1.5 ml dioxane.

The solution was cooled to 8° C., added with 20 μl (0.15 mM)isobutylchlorocarbonate, and caused to react for 35 min.

2. Conjugation with BSA

BSA (Sigma) (210 mg) was solubilised in 5.5 ml water and added with 4 mldioxane and 240 μl 1 N sodium carbonate.

The mixture was cooled to 8° C., added with the whole activatedprogesterone solution (the pH was adjusted to 7.5) and caused to reactfor 30 min. Then, it was added with 25 μl 1 N NaOH and caused to reactfor 4½ hrs.

The solution was dialysed against water overnight.

The pH was brought to 4.5 with 1 M HCl. The precipitate that formed wasallowed to stand at 4° C., recovered by centrifuging, solubilised in 10ml water, while the pH was adjusted to 7.0 with 1 M sodium carbonate,and finally purified by successive passages over acetone (15 ml/passage)at acid pH.

EXAMPLE 9

Salmonella Inactivation.

For the production of antibodies against salmonella, the animal wassensitized using the relevant antigen.

The antigen was produced from the salmonella enteritidis pathogen takenfrom a clinico-pathological material and grown by fermentative route ona medium specific for salmonellas.

The antigen was extracted by bacterial lysis according to methods known(M. Raynaud, A. Turpin, R. Mangalo and B. Bizzini, Croissance ettoxinogenese, Ann. Inst. Pasteur, 1955, 88, 24).

The purification and transformation of same into an immunogen werecarried out by traditional methods (B. Bizzini, A. Turpin and M.Raynaud, Bull. Inst. Pasteur, 1974, 72, 177).

EXAMPLE 10

Animals Immunisation for Polyclonal Antibodies Production

The polyclonal antibodies corresponding to the various immunogenssynthesised in Examples 1 to 9 were produced in the sheep according to amethod already described (cf. A. Johnstone and R. Thorpe,Immunochemistry in Practice, 1982, 27-30, Blackwell Sci. Publ., Oxford).

In particular, the immunisation protocol adopted was as follows:

-   a. animal sensitisation treatment by subcutaneous (sc)    administration of 10 mg immunogen/animal, suspended in 2 ml of a 1:1    (v/v) mixture of PBS, pH 7.4, and Freund complete adjuvant (Sigma    F5881); the sc injection was administered in five different points    (0.4 ml/point) of the animal dorsal region;-   b. booster treatment by intramuscular (im) injection (in the thigh)    of 2.5 mg immunogen/animal, suspended in 1 ml of a 1:1 (v/v) mixture    of PBS, pH 7.4, and Freund's incomplete adjuvant (Sigma F5506);-   c. booster treatments at thirty-day intervals, performed under the    same experimental conditions as described above until antibody    response positivity.

The antibody response positivity was assayed by the ELISA method: themicroplate well was coated with a control immunogen, i.e. an immunogenof the same hapten conjugated with a protein (e.g. HSA; OVA)heterologous in respect of that used for the synthesis of the testimmunogen (product administered). The sensitivity and specificity of theantibodies produced were analysed using a sheep antibody anti-antibodyconjugated with enzyme HRP (Horseradish Peroxidase Sigma A3415);

-   d. the blood for antibodies purification was taken from the animal    jugular on the 15th day following each booster.

EXAMPLE 11

Specific Immunoglobulins (IgGs) Purification

The serum was collected by animal blood centrifugation at 3,000 rpm for15 min; immunoglobulins were precipitated therefrom by treatment with anammonium sulphate saturated solution as described in literature (K.Heide and H. G. Schwick, Salt fractionation of immunoglobulins, inImmunochemistry, vol. 1, 1978, chapter 7, Ed. D. M. Weir, Blackwell Sci.Publ., Oxford).

Excess salts were removed by dialysis against PBS, pH 7.4, in 10 kDcut-off tubing.

The IgGs specific for the hapten (antigen) were separated from the totalimmunoglobulins by affinity chromatography on Sepharose 4B, activatedwith the specific immunogen, as described in literature (S. Fuchs and M.Sela, Immunoadsorbents, Immunochemistry, vol. 1, 1978, chapter 10, Ed.D. M. Weir, Blackwell Sci. Publ., Oxford).

EXAMPLE 12

Specific IgGs Immobilisation on Nitrocellulose Substrate (Sheet)

1. Nitrocellulose Activation

Reaction scheme:Nitrocellulose-OH+H₂C═CH—SO₂—CH═CH₂->Nitrocellulose-O—CH₂—CH₂-SO₂—CH═CH₂

Divinylsulfone (DVS—Sigma V3700) (10 ml) was dissolved in 20 mldimethylformamide and 170 ml 0.5M NaHCO₃/Na₂CO₃ buffer, pH 10. Thenitrocellulose sheet was immersed in said solution where it was kept at21° C. for 10 min, rinsed with distilled water and dried.

The resulting nitrocellulose sheet could be preserved at 4° C. in thedry state for one month at least.

2. Linker Attack to Activated Nitrocellulose

The purpose of said attack is to eliminate the steric hindrance toimmunoglobulins attack.

Reaction scheme:Nitrocellulose-O—CH₂—CH₂—SO₂—CH═CH₂+H₂N—(CH₂)₄—NH₂—->Nitrocellulose-O—CH₂—CH₂—SO₂—CH₂—CH₂—NH—(CH₂)₄—NH₂

The activated nitrocellulose sheet prepared as described above wasimmersed in a 1% (w/v) water solution of 1,4-diaminobutane (Sigma P7505)at 21° C. for 30 min, removed therefrom and washed with distilled water.

3. Immunoglobulins Binding to Cellulose.

Immunoglubulins were bound to the nitrocellulose treated as describedabove by to one of the two following techniques:

3a) IgGs Periodic Oxidation and Binding to the NH₂ Group of the Linker

10 ml of an IgG solution (20 mg/mil) in 0.1M citric acid/Na citratebuffer, pH 5.0, was heated to 37° C. and added with a sodiummetaperiodate solution (NalO₄, 30 mg/ml water) in an IgG/sodiummetaperiodate molar ratio equal to 1:15.

The oxidation was carried out at 37° C. for 5 min, under stirring,sheltered from light, and discontinued by addition of ethylene glycol(Sigma E9129) in a final concentration of 0.01 M.

The linked nitrocellulose sheet, prepared as per point 2, was immersedin 40 ml 1 M Na₂CO₃/NaHCO₃ buffer, pH 10.0, and added with the solutionof oxidised IgGs. The reaction was carried out at 4° C. overnight.

The pH was adjusted to 6.0 by addition of 1 M NaH₂PO₄. Then, a fresh0.26M NaBH₄ solution (10 mg/ml) was added to a final concentration of0.001M. The reduction reaction was carried out at room temperature for30 min.

The nitrocellulose sheet was washed with PBS, pH 7.4, and dried.

3b) Introduction of Aldehydic Groups on Linked Nitrocellulose and IgGsImmobilisation

Reaction scheme:Nitrocellulose-O—(CH₂)₂—SO₂—(CH₂)₂—NH₂+—CHO—(CH₂)₃—CHO->Nitrocellulose-O—(CH₂)₂—SO₂—(CH₂)₂—N═CH—(CH₂)₃—CHO

The nitrocellulose-NH₂ sheet prepared as per point 2 was immersed in a1% (v/v) glutaraldehyde solution in 0.5M NaHCO₃/Na₂CO₃, pH 10, where itwas kept at 21° C. for 15 min, washed with distilled water and dried.

The resulting nitrocellulose sheet could be preserved at 4° C. in thedry state for one month, without any reactivity loss.

The nitrocellulose-CHO sheet was immersed in 100 ml IgGs solution in0.5M NaHCO₃/Na₂CO₃ buffer, pH=10.0, where it was kept at 4° C.overnight.

The sheet was rinsed in PBS, pH 7.4, and immersed in a fresh solution of0.001 M NaBH₄ in PBS, pH 6.0. The reduction reaction was carried out atlaboratory temperature for 30 min. Then, the sheet was rinsed in PBS, pH7.4.

EXAMPLE 13

Specific IgGs Immobilisation of on a Nylon Substrate (Fabric)

Scheme No. 1

1. Nylon Activation

A nylon 6,6[poly(N,N′-hexamethylene adipin diamide—Fluka 74712] fabricwas—immersed in 3.5M HCl where it was kept for 24 hrs, and washed withdistilled water.

Nylon activation by —NH₂ and —COOH reactive groups exposure wasobtained.

2. Conjugation with Immunoglobulins

The immunoglobulins were conjugated with HCl-pretreated nylon accordingto three different procedures:

2a)

Reaction scheme:Nylon-COOH+CMC-<Nylon-COCMC

The —NH₂ groups were blocked by 1-min treatment with concentrated aceticanhydride. The fabric was washed with distilled water first and thenwith 0.1M carbonate buffer, pH 9.5, and finally rinsed in PBS.

The —COOH groups were activated by treatment of the material pretreatedas described above with 4%1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide meta-p-toluenesulfonate(CMC) in water at room temperature for 10 min.

IgGs and nylon-CMC were caused to react in PBS at room temperature for 2hrs and then at 4° C. overnight.

The fabric was washed with PBS, treated with a 2% (w/v) OVA (ovoalbumin)solution In PBS at room temperature, further washed with PBS-Tween 20(0.05%), and finally dried.

2b)

Reaction scheme:Nylon-NH₂+CHO—(CH₂)₃—CHO->Nylon-N═CH—(CH₂)₃—CHO

The nylon fabric, preactivated with HCl, was treated with a 2%glutaraldehyde solution in distilled water at room temperature for 2hrs.

After washing with distilled water, the nylon fabric was treated at roomtemperature for 4 hrs with an IgG solution in PBS in a 1 mg/mlConcentration. The fabric was then washed with PBS, treated with a 2%(w/v) OVA solution in PBS for 1 hr, further washed at room temperaturewith PBS-Tween 20 (0.05%), and dried.

2c)

Reaction scheme:Nylon-NH₂+CH₂═CH—SO₂—CH═CH₂-<Nylon-NH—CH₂—CH₂—SO₂—CH═CH₂

The nylon fabric pretreated with HCl for reactive groups exposure, wasimmersed in 10 ml divinylsulfone (DVS) dissolved in 20 mldimethylformamide (DMF) and 170 ml 0.5M NaHCO₃/Na₂CO₃ buffer, pH 9.0,where it was kept at 21° C. for 1 hr, and washed with distilled water.

100 mg IgGs was dissolved in 20-ml 0.5M NaHCO3/Na₂CO₃ buffer, pH 9.0.The nylon-DVS fabric was immersed in said solution where it was kept atroom temperature overnight.

Once the reaction was completed, nylon was rinsed with PBS and dried.

EXAMPLE 14

Wine Decontamination from Ochratoxin.

The aim of the experiment was to analyse, in parallel, the complexation(elimination) capacity—and relevant rate—of anti-ochratoxin-specificimmunoglobulins (IgGs) for wine decontamination from the toxin. The IgGswere used in the following forms:

-   a. free—as are, not bound to any support;-   b. bound to microspheres made of glass containing primary aminic    groups (Glass-aminopropyl-Sigma G4643-200/400 mesh);-   c. bound to nylon fabric according to the present invention.

Immunoglobulins IgGs were bound to the glass microspheres according tothe following reaction scheme:

5 mg anti-ochratoxin specific IgGs was diluted in 2 ml 0.01M acetatebuffer, pH 5.5, and dialysed against said buffer (purification saltsremoval).

The solution was put in an ice bath, added with 53.5 mg NalO₄, caused toreact under stirring for 20 min, sheltered from light, added with 40 μlethylene glycol, caused to react for 5 min., and dialysed against 0.1 MNa₂CO₃/NaHCO₃ buffer, pH 9.5.

The solution, taken up and brought to 10 ml volume with said buffer, wasadded with 1 g microspheres-NH₂ and caused to react at room temperatureovernight.

The microspheres suspension was added with 800 μl NaBH₄ (4 mg/ml)solution and caused to react at laboratory temperature for 2 hrs.

The microspheres conjugated with IgGs were washed with PBS. Thefollowing conjugation ratio was obtained: 1.84×10⁻⁸ mol IgG/gmicrospheres-NH₂.

Wine decontamination was performed according to the followingexperimental procedure.

The wine used was red type because of its high toxin content (up to 0.4μg/l) according to the state of the art. As revealed by HPLC, theochratoxin content in 300 l wine was 0.265 μg/l.

The wine was subdivided into six 50 l aliquots, which were treated asfollows:

-   aliquot No. 1: under stirring at 150 rpm, with addition of free    anti-ochratoxin IgGs in a ratio of 1 mol IgG:1 mol toxin present;-   aliquot No. 2: under stirring at 150 rpm, with addition of free    anti-ochratoxin IgGs conjugated with glass microspheres in a ratio    of 1 mol IgG:1 mol toxin present;-   aliquot No. 3: under stirring at 150 rpm with addition of    anti-ochratoxin IgGs conjugated to nylon fabric in a ratio of 1 mol    IgG:1 mol toxin present. The fabric used was in the form of strips.    Strips were kept in suspension by ping pong balls at one end and    kept taut, and anyway free to move in the liquid, by weights (lead    sinkers) at the other end.-   Aliquots No. 4 (free IgGs), No. 5 (IgGs on glass microspheres) and    No. 6 (IgGs on nylon fabric) were treated as described above, except    that stirring was performed at 1,000 rpm.

All decontamination experiments were carried out at room temperature.

After 1 h-, 3 h-, and 6 h-contact, a 100 ml quantity was taken from eachwine aliquot and the non-sequestered (free) ochratoxin content wasdetermined.

For the toxin content determination in aliquots Nos. 1 and 4 (withaddition of free IgGs), the analysis was preceded by a dialysis in 10 Kdcut-off tubing to retain the IgG-ochratoxin complexes present.

The following table shows the results obtained under the aforesaidexperimental conditions, expressed as percent abatement of the freetoxin concentration in wine (decontamination capacity of the varioustoxin elimination systems by specific IgGs). % wine toxin abatementafter Method Stirring at 1 h 3 h 6 h Free IgGs  150 rpm 75 90 100 IgGson glass  150 rpm 54 60  62 microspheres IgGs on nylon  150 rpm 95 100 —Free IgGs 1000 rpm 84  95 100 IgGs on glass 1000 rpm 90 100 —microspheres IgGs on nylon 1000 rpm 100 — —The results obtained show that:

-   a. using specific IgGs, wine can be completely decontaminated from    ochratoxin;-   b. using free IgGs, the time taken to complex the whole toxin is    relatively long (probably due to the action of the interfering    substances present in wine);-   c. using IgGs bound to glass microspheres, a fairly vigorous stir is    required to obtain total complexation, likely because, in the    absence of or under gentle stirring, the microspheres tend to    deposit on the bottom. However, vigorous stirring may be    incompatible with the industrial wine-making process;-   d. surprisingly, using IgGs conjugated with the fabric, the whole    toxin present in wine can be complexed within short times and with    low stirring levels completely compatible with the industrial    process.

EXAMPLE 15

Wine Decontamination from Biogenic Amines (Putresceine)

The aim of the experiment was to analyse the complexation (elimination)capacity—and relevant rate—of specific immunoglobulins for winedecontamination from biogenic amine, added extemporaneously to wine inpredetermined concentrations. The IgGs were used in the following forms:

-   a. free—as are, not bound to any support;-   b. immobilised on glass microspheres (Sigma G4663);-   c. immobilised on nylon 6,6 fabric according to the present    invention; and operating under different experimental conditions.

Specific IgGs immobilisation on glass microspheres was performedaccording to the procedure described in Example 14.

The putresceine content in wine was assessed by HPLC according tomethods known.

Also in this case, the wine used was red type. The wine was analysed todetermine the presence, if any, of biogenic amines, then it was added,under stirring, with putresceine up to a final concentration of 4 mg/l.

Wine decontamination was performed according to the followingexperimental procedure.

The wine was subdivided into six 50 l aliquots, which were treated asfollows:

-   aliquot. No. 1: allowed to stand (i.e. without stirring) and added    with free—(non-immobilised) anti-putresceine IgGs in a ratio of 1    mol IgG to 1 mol amine added;-   aliquot No. 2: allowed to stand under the same conditions and added    with anti-putresceine IgGs immobilised on glass microspheres in a    ratio of 1 mol IgG to 1 mol amine added;-   aliquot No. 3: allowed to stand under the same conditions and added    with anti-putresceine IgGs immobilised on nylon fabric according to    the present invention in a ratio of 1 mol IgG to 1 mol amine added.-   Aliquots No. 4 (free IgGs), No. 5 (IgGs immobilised on microspheres)    and No. 6 (IgGs immobilised on nylon fabric) were treated as    described above, except that stirring at 150 rpm was continued    throughout the test.

All decontamination experiments were carried out at room temperature.

After 1 h-, 3 h-, and 6 h-contact, a 100 ml quantity was taken from eachwine aliquot and the putresceine content (non-complexed by more or lessimmobilised specific antibodies) was determined.

For toxin content determination in aliquots Nos. 1 and 4 (treated withfree—non-immobilised—IgGs), the analysis was preceded by a dialysis in100 Kd cut-off tubing to retain the IgG-putresceine complexes present.

The following table shows the results obtained under the aforesaidexperimental conditions, expressed as percent abatement of the freebiogenic amine concentration in wine (decontamination capacity of thevarious amine elimination systems by specific IgGs in different forms).% toxin abatement after Method Stirring 1 h 3 h 6 h Free IgGs no 55 65 80 IgGs on glass no 50 55  58 microspheres IgGs on nylon no 75 95 100Free IgGs 150 rpm 72 87 100 IgGs on glass 150 rpm 65 70  74 microspheresIgGs on nylon 150 rpm 96 100 —The results obtained show that:

-   a. surprisingly, using IgGs immobilised on nylon fabric, food    liquids (wine) can be completely decontaminated also when allowed to    stand, i.e. without stirring procedures, which might be hardly    applicable to production processes;-   b. IgGs immobilised on glass microspheres cannot exert their optimum    decontamination power because, before total complexation, they tend    to deposit on the bottom.

EXAMPLE 16′

Wine Decontamination from Carbamates (Ethyl Carbamate or Urethane)

The capacity of IgGs—either free or variously immobilised on inertsubstrates at different concentration levels—for decontaminating winefrom carbamate, added extemporaneously, was evaluated.

To that purpose, the following specific antiurethane immunoglobulinswere used:

-   a. free—not immobilised on any support;-   b. immobilised on glass microspheres (Sigma G4643) as described in    the preceding examples;-   c. immobilised on nylon 6,6 fabric according to the present    invention.

The ethyl carbamate (urethane) content was determined by gaschromatography according to the method described in literature (H. M.Stahr, Analytical Methods in Toxicology, 1991, p. 0.157, John Wiley andSons, N.Y.).

Wine decontamination was performed according to the following procedure.

A white wine lot was examined to ascertain the presence, if anyl ofethyl carbamate: the quantity found equalled 5 μg/l. The wine wasfurther added with ethyl carbamate up to a final concentration of 500μg/l and then subdivided into four 50 l aliquots, which were treated asfollows:

-   aliquot No. 1: under stirring at 150 rpm, with addition of    free—non-immobilised—anti-urethane IgGs in a ratio of 0.5 mol IgG to    1 mol urethane;-   aliquot No. 2: under stirring at 150 rpm, with addition, as    described in the preceding examples, of anti-urethane IgGs    immobilised on nylon fabric, in a ratio of 0.5 mol IgG to 1 mol    urethane;-   aliquots No. 3 (free IgGs) and No. 4 (IgGs immobilised on nylon    fabric) were treated as described above, but in an IgG/urethane    molar ratio equal to 1:1.

All decontamination experiments were carried out at room temperature.

After 1 h-, 3 h-, and 6 h-contact, a 100 ml quantity was taken from eachwine aliquot and the carbamate content (non-sequestered) in the solutionwas determined.

Also in this case, the aliquots treated with free (non-immobilised) IgGshad been previously subjected to dialysis to retain the IgG-carbamatecomplexes.

The following table shows the results obtained under the aforesaidexperimental conditions, expressed as percent abatement of the chemicalcontaminant concentration in wine. % carbamate IgG/carbamate abatementafter Method ratio by mol 1 h 3 h 6 h Free IgGs 0.5 55 58 68 IgGs on 0.574 90 95 nylon Free IgGs 1 82 90 96 IgGs on 1 94 100 — nylon

Considering that each mol of specific antibody can generally complex twoantigen mols, the above results prove that the IgGs immobilisation onnylon fabric make IgGs surprisingly bioavailable for antigen binding; aproof is that, already at concentrations close to the theoreticalvalues, the contaminant elimination from wine is almost total.

EXAMPLE 17

Milk Decontamination from Aflatoxin

The aim of the experiment was to analyse the capacity—and relevantrate—of anti-aflatoxin specific-IgGs immobilised on different inertsupports for milk decontamination from aflotoxin A1. The IgGs were usedin the following forms:

-   a. anti-aflotoxin specific IgGs Immobilised on glass microspheres    according to the procedure described in the preceding examples;-   b. the same IgGs immobilised on nylon fabric according to the    present invention.

The decontamination experiments were carried out on a sample maintainedat 4° C., i.e. at the usual milk preservation temperature.

Decontamination was performed according to the following experimentalprocedure.

The aflatoxin A1 content in milk was determined by HPLC as known in theart (S. M. Lamplugh, Comparison of three methods for the extraction ofaflatoxins from human serum in combination with a high-performanceliquid chromatographic assay, J. Chromatogr., 1983, 273, 442). Themethod was adjusted according to the nature of the sample.

A milk lot was examined to ascertain the presence of toxin, if any. Thenthe milk was added with aflatoxin up to a final concentration of 0.3μg/l milk and subdivided into 10 l aliquots, which were treated asfollows:

-   aliquot No. 1: under stirring at 150 rpm, with addition of    anti-aflatoxin IgGs immobilised on glass microspheres in a ratio of    1 mol IgG to 1 mol toxin present;-   aliquot No. 2: under stirring at 150 rpm, with addition of    anti-aflatoxin IgGs immobilised on nylon fabric in a ratio of 1 mol    IgG to 1 mol toxin present;-   aliquot No. 3: under stirring at 150 rpm, with addition of    anti-aflatoxin IgGs immobilised on glass microspheres in a ratio of    2 mol IgG to 1 mol toxin present; aliquot No. 4: under stirring at    150 rpm, with addition of anti-aflatoxin IgGs immobilised on nylon    fabric in a ratio of 2 mol IgG to 1 mol toxin present.

In this experiment, it was found it convenient to test higherimmunoglobulins concentrations since the immunologic reactions conductedin the cold are significantly slowed down.

After 1 h-, 3 h-, and 6 h-contact, 100 ml aliquots were taken from themilk and the aflatoxin content (non-sequestered by immunoglobulins) wasdetermined.

The following table shows the results obtained under the aforesaidexperimental conditions, expressed as percent abatement of the freetoxin concentration in milk (decontamination power of the various toxinelimination systems by means of anti-toxin specific IgGs). % aflatoxinIgG:aflatoxin ratio abatement after Method by mol 1 h 3 h 6 h IgGs on1:1 32 45  52 microspheres IgGs on 1:1 65 86 100 nylon IgGs on 2:1 55 78 84 microspheres IgGs on 2:1 80 100 — nylon

The above results prove that the immunoglobulins immobilization on nyloncompared with other immobilization systems—secures a surprisinglyimproved decontamination power. Furthermore, milk contaminants may betotally eliminated by said method also at low temperatures.

EXAMPLE 18

Milk Decontamination from Salmonella Antigens

The aim of the experiment was to evaluate the capacity of specificimmunoglobulins immobilised on an inert substrate for decontaminatingmilk from bacterial contaminants as well as the rationality andsimplicity of the immobilisation methods in view of a successiveapplication to industrial processes.

A further aim of the experiment was to evaluate the suitability ofimmobilised IgGs to be re-used, once adequately washed, in successivetreatments.

To this end, the analysis of milk decontamination from salmonellaantigens was conducted with IgGs in the following forms:

-   a. free—as are, non-immobilised on any support b. immobilized on    glass microspheres (Sigma G4643);-   c. immobilised on nylon fabric according to the present invention.

Milk decontamination was performed according to the followingexperimental procedure.

The presence of salmonella antigens in the food liquid was assayed bycompetitive ELISA, with a specific antibody attached to the microplateand analysis of the competition, for antibody bonding, between theantigen present on the sample and the same antigen conjugated with adetecting enzyme (peroxidase).

A milk lot was added with salmonella antigens up to a finalconcentration of 20 μg/l and then subdivided into 10 l aliquots, whichwere treated as follows:

-   aliquot No. 1: under stirring at 150 rpm, with addition of    free—non-immobilised—salmonella anti-antigen specific IgGs in a    ratio of 1 mol IgG to 1 mol antigen present;-   aliquot No. 2: under stirring at 150 rpm, with addition of    anti-antigen specific IgGs immobilised on glass microspheres in a    ratio of 1 mol IgG to 1 mol antigen present;-   aliquot No. 3 under stirring at 150 rpm, with addition of    anti-antigen specific IgGs immobilised on nylon fabric in a ratio of    1 mol IgG to 1 mol antigen present.

All decontamination experiments were carried out at room temperature.

After 3-h contact, the IgGs were removed from the liquid according tothe following procedure:

-   aliquot No. 1 (free IgGs) by filtration through 0.45 μm membrane;-   aliquot No. 2 (IgGs on glass microspheres) by filtration through    Watmann 1 filter paper;-   aliquot No. 3 (IgGs on nylon) by simple removal of the fabric from    the liquid.

The antigen residue, if any, in milk was measured.

The results expressed as percent abatement of the antigen concentrationin milk (decontamination power of variously immobilised immunoglobulins)are shown in the following table. Method % antigen abatement after 3 hFree IgGs 80 IgGs on glass 72 microspheres IgGs on nylon 100

The above results essentially show the decontamination power of IgGsimmobilised on nylon fabric in respect of other immobilisation systems.

The glass microspheres or the nylon fabric were regenerated by removalof the contaminant bound to the antibodies through a 30-min treatmentwith a 0.1 N HCl solution under gentle stirring. The fabric or themicrospheres were rinsed with PBS and used in successive decontaminationprocesses according to the procedure described above. No experiment wascarried out with non-immobilised IgGs since ‘they are hardly’recoverable.

The following table shows the results obtained in successive treatments;they are expressed as percent abatement of milk contamination fromsalmonella antigen. % antigen abatement after Method 3 treatments 7treatms. 10 treatms. IgGs on glass 67 52 45 microsperhes IgGs on nylon100 100 95

The results obtained show that the immobilisation on nylon surprisinglygives better results than other immobilisation systems, in terms ofdecontaminant regeneration and re-use. It follows that the incidence ofthe decontamination costs on the product cost is considerably reduced.

EXAMPLE 19

Milk Decontamination from Progesterone

The aim of the experiment was to evaluate the capacity of theimmunoglobulins immobilised on an inert support for food liquidsdecontamination from excess steroid hormones.

The experiment was carried out on milk as it contains said hormones invarying amounts depending on the physiological period of milking; by wayof example, the possibility of removing progesterone from milk wasexamined.

The decontamination power of the following IgGs was examined:

-   a. anti-progesterone specific IgGs immobilised on magnetic    microspheres coated with synthetic polymers, suitable for chemical    conjugation;-   b. anti-progesterone specific IgGs immobilised on nitrocellulose    strips according to the procedure of the present invention.

The immobillisation of magnetic microspheres was performed according tothe same procedure as used for the immobilisation on glass.

Milk decontamination was performed according to the followingexperimental procedure.

The progesterone content in milk was evaluated by ELISA, according toknown procedures (J. A. Demetriou, in Meth. in Clin. Chem., 1987, p.253, A. J. Pesce and L. A. Kaplan, Eds., C. V. Mosby Publisher, St.Louis, USA).

Once the progesterone content was checked, milk was subdivided into 10 laliquots, which were treated as follows:

-   aliquot No. 1: use of IgGs immobilised on magnetised microspheres in    such a concentration as to obtain a ratio of 1 mol IgG to 1 mol    progesterone present, under gentle magnetic stirring, which imparts    continuous movement to the microspheres;-   aliquot No. 2: use of IgGs immobilised on nitrocellulose strips in    such a concentration as to obtain a ratio of 1 mol IgG to 1 mol    progesterone, under gentle stirring, which imparts slight movement    to the cellulose strips in the liquid.

All decontamination experiments were carried out at room temperature.

After 1-h and 3-h contact, a 100 ml quantity was taken from each milkaliquot and the progesterone content in the liquid (non-sequestered) wasdetermined.

The microspheres were removed by filtration and the cellulose stripswere removed manually.

The following table shows the results obtained, under the aforesaidexperimental conditions, expressed as percent abatement of theprogesterone concentration in milk (decontamination power ofanti-progesterone specific IgGs immobilised on various inert supports).% progesterone abatement after Method 1 h 3 h IgGs on magnetised 74 88microspheres IgGs on nitrocellulose 97 100

The results point out the decontamination efficiency of IgGs conjugatedto fabrics other than nylon; e.g. nitrocellulose.

They also show that, by operating on large quantities, theimmobilisation on fabric gives significantly better and more profitableresults than the immobilisation on magnetised microspheres.

EXAMPLE 20

Fruit Juice Decontamination from Atrazine

The aim of the experiment was to analyse the capacity of specific IgGsimmobilised on an inert phase for the decontamination of thick foodliquids, such as fruit juices, from chemical decontaminants, such as forexample atrazine.

To this end, anti-atrazine IgGs were immobilised on:

-   a. glass microspheres (Sigma-4643),-   b. nylon fabric, according to the present invention.

Fruit juice decontamination was performed according to the followingexperimental procedure.

The atrazine content in fruit juices was evaluated by gas-chromatographyaccording to common methods (H. M. Stahr, Analytical Methods inToxicology, 1991, pag. 181, John Wiley and Sons, N.Y).

An orange juice as found in commerce was added extemporaneously with anatrazine solution up to a final concentration of 50 μg/l juice.

The juice was subdivided into 5 l aliquots, which were treated asfollows:

-   aliquot No. 1: under stirring at 150 rpm, with addition of    anti-atrazine specific IgGs immobilised on glass microspheres in a    ratio of 2 mol IgG to 1 mol contaminant;-   aliquot No. 2: under stirring at 150 rpm, with addition of    anti-atrazine specific IgGs immobilised on nylon fabric according to    the present invention in a ratio of 1 mol IgG to 1 mol contaminant;-   aliquot No. 3 under stirring at 150 rpm, with addition of    anti-atrazine specific IgGs immobilised on nylon fabric in a ratio    of 2 mol IgG to 1 mol contaminant.

Like in all other experiments, the IgG activated nylon strips wereresuspended in the liquid to be decontaminated by keeping same Insuspension by means of hollow plastic balls at one end of the strip, andtaut, during stirring, by means of small lead sinkers at the other end.

All decontamination experiments were carried out at room temperature.

After 2 and 4 hours, a 100 ml quantity was taken from each juice aliquotand the contaminant atrazine content in the liquid(non-sequestered—free) was determined.

The following table shows the results obtained, under the aforesaidexperimental conditions, expressed as percent abatement of the freeatrazine concentration in the fruit juice (specific IgGs decontaminationcapacity). % atrazine IgG:atrazine abatement after: Method ratio by mol.2 h 4 h IgGs on glass 2:1 45 60 microspheres IgGs on nylon 1:1 65 78IgGs on nylon 2:1 87 95

These results show that also “thick” food liquids can be decontaminated,i.e. under the particular conditions of the antibody/substance to theeliminated reaction medium.

They also show that when, in the decontamination, the fabrics are usedas an antibodies immobilisation means, the reaction conditions andconsequently the decontamination capacity are significantly improved.

1-26. (canceled)
 27. Process for decontaminating a liquid food from oneor more chemical and/or biological contaminants wherein one or morebiocompatible polymer membranes to which antibodies specific for saidcontaminants are chemically conjugated through a linker, are immersedinto said liquid.
 28. The process according to claim 27, wherein thebiocompatible polymer is selected from: nylon, cellulose, polyacrylate,polyester or viscose, their derivatives or mixture thereof.
 29. Theprocess according to claim 28, wherein said polymer is nylon.
 30. Theprocess according to claim 28, wherein said polymer is in the form ofwoven non woven fabric.
 31. The process according to claim 27, whereinthe linker is selected from the group consisting of:CH₂—CH₂—SO₂—CH₂-—H₂—NH—(CH₂)₄—N═CH—(CH₂)₃—CH═O or a peptide comprising adiamino-monocarboxylic amino acid or a monoamino-dicarboxylic aminoacid.
 32. The process according to claim 31, wherein thediamino-monocarboxylic amino acid is chosen between arginine and lysineand the monoamino-dicarboxylic amino acid is chosen between glutamicacid and aspartic acid.
 33. The process according to claim 27, whereinsaid contaminants are selected from the group consisting of:parasiticides, weed killers, pesticides, drugs and metabolites thereof,hormones and metabolites thereof, wine malolactic fermentation products,and toxins.
 34. The process according to claim 33, wherein saidcontaminants are further selected from the group consisting of:atrazine, aflatoxin, ochratoxin, fumonisine, cadaverine, putresceine,urethane, progesterone and salmonella antigen.
 35. The process accordingto claim 27, wherein said membranes are kept immersed in the liquid fora time from 1 to 24 hours.
 36. The process according to claim 27,wherein said membranes are kept immersed in the liquid for a time from 1to 6 hours.
 37. The process according to claim 27, which is performedwithout stirring.
 38. The process according to claim 27, wherein saidfood liquid is selected from the group consisting of: wine, milk, fruitjuice, vegetable juice, beer and water.
 39. The process according toclaim 27, wherein said antibodies are polyclonal antibodies.
 40. Theprocess according to claim 27, wherein the total surface of themembrane/s for contaminant is such that the molar ratio of theimmobilized antibody to the contaminant toward which the antibody isdirected is
 1. 41. The process according to claim 40, wherein the saidmolar ratio is from 1 to
 5. 42. The process according to claim 41,wherein said molar ratio is from 1 to 2.